The major goal of this research is to understand how the eucaryotic centromere DNA and proteins assemble into a kinetochore complex that associates with a spindle fiber to ensure proper chromosome segregation. Our research focuses on four main goals: 1) analysis of the centromere DNA sequences required for proper chromosome segregation 2) investigation of the centromeric chromatin structure, 3) study of centromeric binding to the nuclear scaffold and nuclear membrane, and 4) characterization of the kinetochore components. Mutagenesis studies of centromere DNA have delineated the roles played by different CEN DNA elements in mitotic and meiotic chromosome segregation. Current analysis of completely synthetic centromeres has shown that the arrangements of the symmetrical CEN sequence elements are important for centromere function. We have used nuclease mapping to show that mutant CEN DNAs can assemble into aberrant centromeric chromatin structures. We are now extending this work using in vivo footprinting to analyze the CEN DNA:protein interactions at the nucleotide level. Specific bases in conserved centromere sequences are in contact with proteins in vivo. Comparison of wildtype and mutant protection patterns and analysis of the footprint pattern in cells during the cell cycle are in progress. Modifications of this approach will be used to study the conformation of the DNA helix at the centromere. Yeast centromere DNA is bound to the nuclear scaffold, a structure that contains topoisomerase II. Yeast centromeres may also associate with the nuclear membrane. The importance of these interactions will be investigated using our library of CEN DNA mutants. These studies will help to clarify the role of the centromere in chromosome structure and organization at all times during the cell cycle. Biochemical and genetic approaches are underway to characterize the components of the kinetochore. A major effort involves a novel genetic screen we have developed to isolate years mutants defective in chromosome segregation. A chromosome with a mutant centromere is used to promote detection of trans-acting mutants defective in centromere function. Some of these mutants exhibit allele specificity, compelling evidence that these mutations affect components of the centromere. We are currently cloning the CND genes and will raise antisera against these proteins for use in experiments designed to investigate the functions of the CND proteins.